Vol. 173, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 276-282

November 30, 1990

EFFECTS

OF

HERBIMYCIN

A

AND

KINASE

Hidesuke

Department

Fukazawa,

VARIOUS

ACTIVITY

Satoshi

of A n t i b i o t i c s , Kamiosaki,

SH-REAGENTS

ON

p60 v-src

IN V I T R O

Mizuno

National

and Y o s h i m a s a

Institute

Shinagawa-ku,

Tokyo

Uehara

of Health,

141,

2-10-35

Japan

Received October 5, 1990 Summary H e r b i m y c i n A is an a n t i b i o t i c w h i c h r e v e r s e s transf o r m a t i o n c a u s e d by src f a m i l y o n c o g e n e s . It i n a c t i v a t e s p60 vsrc in vitro, p o s s i b l y b y b i n d i n g to r e a c t i v e S H - g r o u p ( s ) of the kinase. We e x a m i n e d e f f e c t s of v a r i o u s S H - r e a g e n t s on p60 v-src and o b s e r v e d that N - [ p - ( 2 - b e n z i m i d a z o l y l ) p h e n y l ] m a l e i m i d e (BIPM) or N - ( 9 - a c r i d i n y l ) m a l e i m i d e (NAM) w e r e p o t e n t i n a c t i v a t o r s of the kinase, whereas N-ethylmaleimide (NEM) required high concentrations, and -- i o d o a c e t a m i d e was total ly i n e f f e c t i v e in r e d u c i n g the k i n a s e activity. Pretreatment of p60 v - s r c i m m u n e c o m p l e x w i t h N E M and i o d o a c e t a m i d e , however, p r o t e c t e d the k i n a s e from i n a c t i v a t i o n by h e r b i m y c i n A, BIPM, and NAM. The r e s u l t s suggest that SH-group(s) to w h i c h h e r b i m y c i n A binds is n o t essential for t h e k i n a s e activity, b u t is p o s i t i o n e d in t h e v i c i n i t y of the a c t i v e center. ©1990Academic P..... Inc.

Transformation the

product

kinase

of

in

essential

and

the

to

I ).

reverse I ), a

inactivation phenotypes

to

the

v-src

for

(I).

both

course we

thereby (2,

of

the

The

is

of

tyrosine

plasma

membrane

and

activity maintenance

screening

found

by

a

kinase

that

for

is of

agents

herbimycin

antibiotic,

reversing 3

is m e d i a t e d

which

initiation

ansamycin

states

virus

p60 v - s r c

transformation,

p60 v-src,

normal

face

of

In the

benzoquinonoid

sarcoma

p60 v-src,

cytoplasmic

Expression

state

of

by R o u s

oncogene,

sufficient

transformed

which (Fig

v-src

attached

(reviewed

of cells

induced

various

transformed

antibiotic

inactivated

Abbreviations: BIPM, N - [ R - ( 2 - b e n z i m i d a z o l y l ) p h e n y l ] m a l e i m i d e ; DMSO, d i m e t h y l sulfoxide; Hepes, N-(2-hydroxyethyl)piperazine-N_~'2 - e t h a n e s u l f o n i c acid; IC50 , c o n c e n t r a t i o n w h i c h r e d u c e s the a c t i v i t y to 50% of the control; Mes, 2 - ( N - m o r p h o l i n o ) e t h a n s u l f o n i c acid; NAM, N - ( 9 - a c r i d i n y l ) m a l e i m i d e ; NEM, Nethylmaleimide; TBR, t u m o r - b e a r i n g rabbit.

0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

276

A

Vol. 173, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

O

H3C

H3

N H

f7 3.2.oc0 '

CH 3 CH 3 0 C H 3 Figure I. Structure of herbimycin A.

p60 v-src group(s) the

kinase

of the kinase

herbimycin

effects

activity

A mode

on the kinase

in

(4). of

vitro,

possibly

In order

p60 v-src

with those

MATERIALS

by

to gain further

inactivation,

of known

AND

binding

we

to

insight compared

SHinto its

SH-reagents.

METHODS

Materials H e r b i m y c i n A was isolated as described previously (2). BIPM (5) was purchased from Kanto Chemicals Co., Tokyo, Japan, NAM (6) from Dojin Laboratories, Kumamoto, Japan, NEM from Nacalai Tesque, Kyoto, Japan, and iodoacetamide from Wako Pure Chemical Industries, Ltd., Osaka, Japan. TBR serum (#I011) and monoclonal antibody 327 were products of T r a n s f o r m a t i o n Research, Inc., Framingham, MA, and Oncogene Science, Inc. , M i n e o l a , NY, respectively. Formalin-fixed Staphylococcus aureus (Pansor~n) was a product of Calbiochem-Behring, La Jolla, CA. [gamma-°~P] ATP was obtained from ICN Radiochemicals, Irvine, CA. Methods NIH/3T3 cells infected with Schmidt R u p p i n - D strain of Rous sarcoma virus were lysed in 20mM Hepes, pH 7.4, ImM EDTA, 0.1mM Na3VO4, 150mM NaCI, and I% Triton X-100 containing 25~g/ml each of protease inhibitors p h e n y l m e t h y l s u l f o n y l fluoride, antipain, leupeptin, and pepstatin A. The lysate was centrifuged at 15,000 x g for 10 min, and the supernatant was incubated with TBR serum 1011 or with monoclonal antibody 327 plus rabbit anti-mouse IgG. The immune-complexes were collected onto formalin-fixed Staphylococcus aureus, washed, and suspended in 20mM Hepes, pH 7.4 (for treatment with h e r b i m y c i n A or iodoacetamide) or 20mM Mes, pH 6.0 (for treatment with BIPM, NAM, or NEM). To a 90~i suspension, I0~i of SH-reagent dissolved in DMSO was added, and the mixture was incubated at 25°C for 30 min. The immune-complex was w a s h e d and assayed for k i n a s e activity as p r e v i o u s l y described (4), except that I mM d i t h i o t h r e i t o l was included in the reaction mixture.

RESULTS

Effects

of SH-inhibitors

We examined BIPM,

and

NAM

effects on

on p60 v-src

kinase

of four SH-reagents,

p60 v-src

kinase 277

iodoacetamide,

activity

in

vitro.

NEM, The

Vol. 173, No. 1, 1990 structures

of the

reduced

the

IC50

about

of

compounds

BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS four

TBR

IgG

IUM

also

more

60%

of

(Fig

concentration (data

not

of 4,

to

TBR

autophosphorylating immunoprecipitates,

complexes

with

to

minimize

at

pH

7.4

reagent,

gave

heavy

was

but

0

still

remained

the

to in

with

results was

never

(data

totally

not

the

10mM

NEM the

exceeded

45%

destroy

the 327

required

high

the

performed

at

but

shown).

ineffective

immunepH

6.0

treatments Another

SH-

in

reducing

10

1 0.1 0.01 ~M

H 0

BIPM NEM

100

NAM 10

1

0.1 0.01 ,uM

0

100

94--> 67--, 43--,

I~]]'"NH20 ~